Non-porous film for culturing cells

ABSTRACT

The invention relates to collagenous polypeptide films on which cells are cultivated. In particular the invention relates to such films that are used to treat wounds such as severe burns or physical or chemical injury. The invention also related to methods for producing such films.

FIELD OF THE INVENTION

The invention relates to gelatin comprising films on which cells can beor are cultivated. In particular the invention relates to such filmsthat are used to treat wounds, such as severe burns or physical orchemical injury of the skin or wounds caused by diseases. The inventionalso related to methods for producing such films and the use of suchfilms. In a further aspect of the invention human artificial skin grownon such films are provided, and methods of making these.

BACKGROUND

Films on which cells are cultured are used in the treatment of skinwounds such as for example wounds caused by severe burns or mechanicalor chemical injuries or in diseases where extensive loss of skinoccurred. In cases of acute extensive skin loss treatment generallyinvolves two phases. In the first phase the requirements for a filmmaterial are directed towards short term requirements such ascontrolling moisture flow through the wound and shielding frominfectious agents. In the second stage long term effects are consideredsuch as non-antigenicity, and skin regeneration.

Development of such materials is in the direction of multilayermaterials of increasing complexity as described in, for example, EP 0686 402, WO 03/101501. Many patent applications disclose the use ofporous collagen or gelatin matrices or sponges that require theformation of collagen fibrils and forming of a porous network, forexample by freeze drying, before crosslinking the porous matrices as infor example EP 0 177 573, EP 0 403 650, EP 0 403 650, EP 0 411 124, EP 0702 081, U.S. Pat. No. 4,016,877 and U.S. Pat. No. 4,294,241.

In applications for wound treatment fibroblast and keratinocyte layersare cultured on a collagen or gelatin matrix. In such cultures thefibroblast cells and/or keratinocyte cells are usually actually embeddedin the matrix material, due to the porous nature of the collagen orgelatin matrix. EP 0 243 132 describes culturing of fibroblasts on aninsoluble collagen film and the subsequent culturing of keratinocytes ontop of the fibroblast layer, but has as a drawback that the fibroblastand keratinocyte layers are in contact.

WO 80/01350 discloses production of a living tissue by culturingkeratinocytes on a collagen layer in which fibroblasts are imbedded, butthis also means that the fibroblast layer and the keratinocyte layersare in contact.

WO 91/16010 describes a complex material based on a non-porous collagengel that is stabilized by iodine and which is laminated on top of aporous collagen sponge containing fibroblasts. Keratinocytes arecultured on top of the stabilized collagen gel. The porous collagensponge is crosslinked to prevent too fast biodegradation.

Use of recombinant collagen or gelatin is disclosed in e.g. WO 00/09018but describes the formation of crosslinked sponges of collagen fibrils.WO 04/78120 also discloses porous structures from recombinant collagen.

Films are also used to test for example allergic reactions to topicalapplications comprising medicines, pharmaceuticals or cosmetics.

In spite of the above described materials there remains a need foralternative films for culturing cells that are suitable for treatment ofwounds involving the loss of skin.

SUMMARY OF THE INVENTION

It is an object of the invention to provide a film suitable for making ahuman artificial skin equivalent and a method for making such a skinequivalent. It is especially an object to provide a film which enablesrapid growth of autologous cells on it surfaces and which enhances cellattachment (adhesion) to the film and cell-to-cell binding of theautologous cells. More specifically it is an object to provide a fullequivalent of human skin. It is further an object of the invention toprovide a method to produce such a film cheap and efficiently with highspeed.

Thus it is an object of this invention to provide a non-antigenic filmsuitable for culturing human and/or mammalian cells of which thebiodegradability can be regulated, and particularly it is an objectiveto provide such a film that is permeable to molecules, includingpolypeptides and proteins, of up to 25 kilo Dalton.

It is also an objective of the invention to provide a film which issuitable as a test substrate for medicines, pharmaceuticals orcosmetics. In particular the effect of compounds contacted with livingor viable cells present on the film can be tested.

Surprisingly it was found that all these objectives were met by anon-porous film comprising on at least one side thereof a layercomprising living or viable cells and wherein the non-porous filmcomprises a collagenous polypeptide comprising at least one GXY domainhaving a length of at least 5 consecutive GXY triplets, wherein X and Yeach represent any amino acid and wherein at least 20% of the aminoacids of said collagenous polypeptide are present in the form ofconsecutive GXY triplets, characterized in that the film thickness whenplaced in demineralized water of 37 degrees Celsius for 24 hours is atmost 10 times its initial thickness.

The non-porous film preferably comprises a collagenous polypeptidecomprising at least one GXY domain having a length of at least 5consecutive GXY triplets, wherein X and Y each represent any amino acidand wherein at least 20% of the amino acids of said collagenouspolypeptide are present in the form of consecutive GXY triplets,characterized in that said film is crosslinked by adding between 0.02millimol and 5.0 millimol of a crosslinking compound per gramcollagenous polypeptide.

DESCRIPTION OF THE INVENTION

When extensive skin loss occurs, wounds are generally treated in twophases. There remains a need for films that can specifically be matchedto the requirements of each treatment phase or more specifically, to thedesired biodegradation speed while being sufficiently permeable tocompounds that are involved in culturing cells on the film, specificallyfor compounds that promote growth of cells on both sides of the film.

The present inventors found that such films can be made which arematched to the requirements of each treatment phase by careful choice ofthe swelling behavior and permeability. Swelling behavior is theincrease of the initial thickness of a non-porous collagenouspolypeptide film when placed in a liquid. In the art it is taught that apore size of at least 1 micron is necessary to provide enoughpermeability for compounds involved in wound healing such as nutrientsand growth factors, especially when fibroblasts and keratinocytes arepresent within a matrix or in different matrices, see for example EP-0702 081 and also the reference in column 4, lines 44-49 therein. Thepresent inventors found, however, that films that are non-porous, orhave an average pore-size of less than 1 micron, are capable of takingup water and are permeable for compounds involved in wound healing.Although it was recognized in prior art as early as 1976, or evenearlier, that crosslinking is necessary to prevent too fastbiodegradation, it was not recognized until now that the degree ofcrosslinking can be advantageously used to adjust swellability and thusbiodegradability and permeability. Thus the use of films of thisinvention for the preparation of a composition for treating wounds is anaspect of this invention. The films of this invention can be used tomatch any particular treatment, especially first phase or second phasetreatment, by providing a non-porous film comprising on at least oneside thereof a layer comprising living or viable cells and wherein thenon-porous film thickness increases up to 10 times its initial thicknesswhen placed in demineralized water at 37 degrees Celsius. Depending onthe desired permeability or biodegradability the film swells at most 8times, or at most 6 times or at most 4 times its original thickness inwater. Preferably the film swells at least 2 times its originalthickness in water. In particular, a method was developed to producenon-porous films having a desired degree of cross-linking and thereforealso a desired biodegradation speed and permeability in vivo (aftercontact with skin wounds e.g. during treatment phase one or two). In oneembodiment of the invention films with a desired, predetermined degreeof cross linking are non-porous films (suitable for cultivating livingor cells on at least one side thereof) comprising a collagenouspolypeptide that comprises at least one GXY domain having a length of atleast 5 consecutive GXY triplets, wherein X and Y each represent anyamino acid and wherein at least 20% of the amino acids of saidcollagenous polypeptide are present in the form of consecutive GXYtriplets. In one embodiment the films according to the invention arecrosslinked by adding one or more crosslinking compounds in an amount ofbetween about 0.02 and 5.0 millimol per gram collagenous polypeptide,preferably between 0.1 to 1.0 millimol/g. In another embodiment nocross-linking compound is present, but the (equivalent) degree ofcross-linking is achieved by radiation. In yet another embodimentcross-linking is achieved by a combination of radiation and addition ofone or more cross-linking compounds.

A further advantage of the method and films according to the inventionis, that the step of forming fibrils, which is necessary when makingporous structures is now obsolete. Further, also the step of freezedrying which is involved in obtaining a porous material is now obsolete,(although both steps may still optionally be carried out) therebyreducing the time and energy that is involved in producing artificialskin and making it possible to produce the non-porous film of thisinvention efficiently and with high speed.

The term “non-porous” means that essentially no micropores are formed asin for example EP 0 177 573, EP 0 403 650, EP 0 403 650, EP 0 411 124,EP 0 702 081, U.S. Pat. No. 4,016,877 or U.S. Pat. No. 4,294,241. Theterm ‘porous’ or ‘microporous’ can be ambiguous, and one may define acrosslinked collagen or gelatin layer as being ‘porous’ on a nano-scale.In the broadest sense, non-porous means in this case that the averagepore-size is less than 1 micron, as determined by scanning electronmicroscopy (SEM) described in for example by Dagalakis et. al. (Designof an artificial skin Part III Control of pore structure—Journal ofBiomedical Materials Research, Vol. 14, 519 (1980)).

The non-porous film is however permeable for molecules, includingpolypeptides or proteins, of up to 5 kilo Dalton, preferably up to 10kilo Dalton and more preferably up to 25 kilo Dalton. In comparison to aglobular protein, permeability of a linear protein such as for example acollagen may be higher, up to between 30 and 40 kilo Dalton.

In one embodiment crosslinking of the collagenous polypeptide isachieved by addition of one or more crosslinking agents. These compriseagents that start crosslinking spontaneously upon addition tocollagenous polypeptide solution, or after adjusting for example, pH, orby photo initiation or other activation mechanisms. In this particularembodiment a number of millimol crosslinks per gram collagenouspolypeptide is defined as being equal to the amount of crosslinkingagent that has reacted with the collagenous polypeptide.

In another embodiment crosslinking of the collagenous polypeptide isachieved by exposure to radiation such as UV-radiation or electron beam.In this particular embodiment of the invention a number of millimolcrosslinks is defined as the amount of crosslinking agent that wouldneed to be added to obtain the same number of crosslinks as are obtainedby exposure to radiation.

In other words, the exposure to radiation results in an equivalentdegree of cross-linking as the addition of between about 0.02 millimolto about 5.0 millimol of crosslinking compound per gram collagenouspolymer does. The amount of crosslinking agent to be added to obtain acertain number of crosslinks can be calculated or determinedexperimentally. In case of exposure to radiation the required exposuretime and intensity has to be determined experimentally, but this iswithin the capability of a skilled person without undue burden. Thedegree of crosslinking can be determined in several ways. In one method,the degree of swelling of the crosslinked collagenous polypeptide ismeasured by soaking the film in demineralized water and measuring theincrease in thickness (swelling) or the increase in weight resultingfrom water uptake. A series of radiation exposures is then compared to aseries in which varying amounts of crosslinking agent is added.Comparing the results of a swelling test provides a correlation betweenthe two methods of crosslinking. A method for measuring swelling ofcollagenous films is described for example by Flynn and Levine (Photogr.Sci. Eng., 8, 275 (1964).

Suitable crosslinking agents are preferably those that do not elicittoxic or antigenic effects when released during biodegradation. Suitablecrosslinking agents are, for example, one or more of glutaraldehyde,water-soluble carbodiimides, bisepoxy compounds, formalin,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, N-hydroxy-succinimide,glycidyl ethers such as alkylene glycol diglycidyl ethers orpolyglycerol polyglycidyl ether, diisocyanates such as hexamethylenediisocyanate, diphenylfosforylazide, D-ribose. Crosslinking techniquesare also described by Weadock et. al. in Evaluation of collagencrosslinking techniques (Biomater. Med. Devices Artif. Organs,1983-1984, 11 (4): 293-318). In a preferred embodiment water-soluble1-ethyl-3-(3-dimethylaminopropyl) carbodiimide is used.

In one embodiment the film is particularly suitable for the first phasetreatment and is crosslinked by adding between 0.02 and 1.0 millimolcrosslinking compound(s) per gram collagenous polypeptide (or radiationinduced crosslinking which is equivalent hereto). Thus, thecross-linking compound(s) may be present in an amount of about 0.02,0.05, 0.1, 0.25, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 millimol/grampolypeptide.

In another embodiment the film is particularly suitable for second phasetreatment and is crosslinked by adding between 0.5 and 5.0 millimolcrosslinking compound(s) per gram collagenous polypeptide (or radiationinduced crosslinking which is equivalent hereto), preferably about 1.0to 2.5 millimol/g. Thus, the cross-linking compound(s) may be present inan amount of about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0 and 5.0millimol/gram polypeptide.

In yet another embodiment the film can be used as an intermediatebetween first and second phase treatment and is crosslinked by addingbetween 0.25 and 2.5 millimol crosslinking compound(s) per gramcollagenous polypeptide (or radiation induced crosslinking which isequivalent hereto).

Another way to express the amount of crosslinking agent is the molarratio with lysine residues in the polypeptide. Especially in case ofrecombinantly produced collagenous polypeptides the number of lysineresidues can be increased as desired. Many crosslinking agents bind tolysine residues and/or N-terminal amines. Natural gelatin containsbetween 25 and 27 lysines per 1000 amino acids. In recombinant collagensor collagenous polypeptides this can be reduced to for example equal toor less than about 20, 15, 10 or 5 lysines per 1000 amino acids orincreased to for example equal to or more than about 30, 40 or 50lysines per 1000 amino acids.

For example, a recombinant collagen-like polypeptide monomer isdescribed in EP1 398 324 that contains 8 lysines in a sequence of 204amino acids, or 39 lysines per 1000 amino acids. Preferably thenon-porous films according to the invention comprise between 0.01 and12.5 millimol crosslinking compound(s) per millimol lysine in thecollagenous polypeptide, or between 0.1 and 10 millimol per millimollysine or between 1 and 5 millimol per millimol lysine, depending on theamount of lysines present in the collagenous polypeptide.

Suitable collagenous polypeptides to make the films according to theinvention are collagens or gelatins from natural, synthetic orrecombinant sources or mixtures thereof. Although strictly speakingthere is a difference between collagen and gelatin, these differencesare in principle not essential to the invention, although specificrequirements may make the selection of collagen or gelatin for a certainapplication obvious. In this respect “collagen” may also be read as“gelatin” and “collagenous polypeptide” may also be read as “gelatinouspolypeptide”. A collagenous or gelatinous polypeptide is thus defined asbeing a polypeptide in which at least one GXY domain is present of atleast a length of 5 consecutive GXY triplets and at least 20% of theamino acids of the collagenous polypeptide are present in the form ofconsecutive GXY triplets, wherein a GXY triplet consists of Grepresenting glycine and X and Y representing any amino acid. Suitablyat least 5% of X and/or Y can represent proline and in particular atleast 5%, more in particular between 10 and 33% of the amino acids ofthe GXY part of the collagenous polypeptide is proline. Collagenouspolypeptides which can be obtained from natural gelatin can be forexample alkaline processed gelatin, acid processed gelatin, hydrolyzedgelatin or peptized gelatin resulting from enzymatic treatment. Naturalsources can be the skin or bones of mammals such as cattle or pigs butalso of cold-blooded animals such as fish.

The collagenous polypeptide preferably has an average molecular weightof less than 150 kilo Dalton, preferably of less than 100 kilo Dalton.Ranges of between 50 an 100 kilo Dalton are suitable or hydrolyzedcollagenous polypeptides of less than 50 kilo Dalton or between 5 and 40kilo Dalton may be used. Preferably the collagenous polypeptides have anaverage molecular weight of at least 5 kilo Dalton, preferably at least10 kilo Dalton and more preferably of at least 30 kilo Dalton. A smalleraverage molecular weight means that more crosslinking compound(s) shouldbe added to obtain a certain permeability than with larger polypeptides.However, lower molecular weights may be preferred for example inproduction of the non-porous film where lower molecular weight has alower viscosity which makes higher concentrations of collagenouspolypeptides possible.

The method of making recombinant collagenous polypeptides has beendescribed in detail in patent applications U.S. Pat. No. 6,150,081 andUS 2003/229205 by the same applicant, the content of which is hereinincorporated by reference. The methodology is described in thepublication ‘High yield secretion of recombinant gelatins by Pichiapastoris’, M. W. T. Werten et al., Yeast 15, 1087-1096 (1999).

Recombinantly produced collagenous polypeptides are preferred becausethe detrimental effects involved in using gelatin or collagen fromanimal sources, such as for example BSE, are avoided. Also, bettercontrol of parameters such as size distribution, amino acid sequence orthe occurrence of specific amino acids is possible. Preferably suchrecombinant collagenous polypeptides have even lower antigenicity thannatural gelatins.

In one embodiment the recombinant collagenous polypeptide does not formstable triple helices, specifically not at temperature of more than 5degrees Celsius, or at temperatures higher than 25 degrees Celsius. Suchcollagenous polypeptides have preferably an amount of prolines presentin GXY triplets that is comparable to collagen originating from mammalsor collagens originating from cold-blooded animals such as fish. Toprevent stable triple helix formation less than 2 number percent,preferably less than 1 number percent, of the amino acids present in thecollagenous polypeptide are hydroxylated. Occurrence of hydroxyprolinescan be reduced to be practically zero by expression in micro organismsthat do not co-express a prolylhydroxylase or fulfill that function inanother way. Practically zero means that the presence of hydroxyprolinesin the growth medium of for example yeasts may result in incorporationof some of these amino acids into the collagenous polypeptide.Recombinant collagen-like polypeptides that are not hydroxylated andhave the advantage of avoiding the occurrence of anaphylactic shock aredescribed in EP 1 238 675.

In a preferred embodiment the non-porous film comprises collagenouspolypeptides with excellent cell attachment properties, and which do notdisplay any health related risks, by production of RGD-enrichedcollagenous polypeptides in which the percentage of RGD motifs relatedto the total number of amino acids is at least 0.4. If the RGD-enrichedcollagenous polypeptide comprises 350 amino acids or more, each stretchof 350 amino acids contains at least one RGD motif. Preferably thepercentage of RGD motifs is at least 0.6, more preferably at least 0.8,more preferably at least 1.0, more preferably at least 1.2 and mostpreferably at least 1.5.

A percentage RGD motifs of 0.4 corresponds with at least 1 RGD sequenceper 250 amino acids. The number of RGD motifs is an integer, thus tomeet the feature of 0.4%, a collagenous polypeptide consisting of 251amino acids should comprise at least 2 RGD sequences. Preferably theRGD-enriched recombinant collagenous polypeptide comprises at least 2RGD sequence per 250 amino acids, more preferably at least 3 RGDsequences per 250 amino acids, most preferably at least 4 RGD sequencesper 250 amino acids. In a further embodiment an RGD-enriched collagenouspolypeptide comprises at least 4 RGD motifs, preferably 6, morepreferably 8, even more preferably 12 up to and including 16 RGD motifs.

The term ‘RGD-enriched collagenous polypeptide’ in the context of thisinvention means that the collagenous polypeptides have a certain levelof RGD motifs, calculated as a percentage of the total number of aminoacids per molecule and a more even distribution of RGD sequences in theamino acid chain than a natural gelatin. In humans up to date 26distinct collagen types have been found on the basis of protein and orDNA sequence information (see K. Gelse et al, Collagens-structure,function and biosynthesis, Advanced Drug Delivery reviews 55 (2003)1531-1546). Sequences of natural gelatins, both of human and non-humanorigin, are described in the Swiss-Prot protein database. Here belowfollows a list of suitable human native sequences, identified by theirentry name and primary accession number in the Swiss-Prot database, thatmay serve as a source of parts of natural sequences comprised in theRGD-enriched collagenous polypeptides comprised in the non-porous filmsof this invention.

CA11_HUMAN (P02452) Collagen alpha 1(1) chain precursor. {GENE:COL1A1}—Homo sapiens (Human)

CA12_HUMAN (P02458) Collagen alpha 1(II) chain precursor [Contains:Chondrocalcin]. {GENE: COL2A1}—Homo sapiens (Human)

CA13_HUMAN (P02461) Collagen alpha 1(III) chain precursor. {GENE:COL3A1}—Homo sapiens (Human)

CA14_HUMAN (P02462) Collagen alpha 1(IV) chain precursor. {GENE:COL4A1}—Homo sapiens (Human)

CA15_HUMAN (P20908) Collagen alpha 1(V) chain precursor. {GENE:COL5A1}—Homo sapiens (Human)

CA16_HUMAN (P12109) Collagen alpha 1(VI) chain precursor. {GENE:COL6A1}—Homo sapiens (Human)

CA17_HUMAN (Q02388) Collagen alpha 1(VII) chain precursor (Long-chaincollagen) (LC collagen). {GENE: COL7A1}—Homo sapiens (Human)

CA18_HUMAN (P27658) Collagen alpha 1(VIII) chain precursor (Endothelialcollagen). {GENE: COL8A1}—Homo sapiens (Human)

CA19_HUMAN (P20849) Collagen alpha 1(IX) chain precursor. {GENE:COL9A1}—Homo sapiens (Human)

CA1A_HUMAN (Q03692) Collagen alpha 1(X) chain precursor. {GENE:COL10A1}—Homo sapiens (Human)

CA1B_HUMAN (P12107) Collagen alpha 1(XI) chain precursor. {GENE:COL11A1}—Homo sapiens (Human)

CA1C_HUMAN (Q99715) Collagen alpha 1(XII) chain precursor. {GENE:COL12A1}—Homo sapiens (Human)

CA1E_HUMAN (P39059) Collagen alpha 1(XV) chain precursor. {GENE:COL15A1}—Homo sapiens (Human)

CA1F_HUMAN (Q07092) Collagen alpha 1(XVI) chain precursor. {GENE:COL16A1}—Homo sapiens (Human)

CA1G_HUMAN (Q9UMD9) Collagen alpha 1(XVII) chain (Bullous pemphigoidantigen 2) (180 kDa bullous pemphigoid antigen 2). {GENE: COL17A10RBPAG2 OR BP180}—Homo sapiens (Human)

CA1H_HUMAN (P39060) Collagen alpha 1(XVIII) chain precursor [Contains:Endostatin]. {GENE: COL18A1}—Homo sapiens (Human)

CA1I_HUMAN (Q14993) Collagen alpha 1(XIX) chain precursor (Collagenalpha 1(Y) chain). {GENE: COL19A1}—Homo sapiens (Human)

CA21_HUMAN (P08123) Collagen alpha 2(I) chain precursor. {GENE:COL1A2}—Homo sapiens (Human)

CA24_HUMAN (P08572) Collagen alpha 2(IV) chain precursor. {GENE:COL4A2}—Homo sapiens (Human)

CA25_HUMAN (P05997) Collagen alpha 2(V) chain precursor. {GENE:COL5A2}—Homo sapiens (Human)

CA26_HUMAN (P12110) Collagen alpha 2(VI) chain precursor. {GENE:COL6A2}—Homo sapiens (Human)

CA28_HUMAN (P25067) Collagen alpha 2(VIII) chain precursor (Endothelialcollagen). {GENE: COL8A2}—Homo sapiens (Human)

CA29_HUMAN (Q14055) Collagen alpha 2(IX) chain precursor. {GENE:COL9A2}—Homo sapiens (Human)

CA2B_HUMAN (P13942) Collagen alpha 2(XI) chain precursor. {GENE:COL11A2}—Homo sapiens (Human)

CA34_HUMAN (Q01955) Collagen alpha 3(IV) chain precursor (Goodpastureantigen). {GENE: COL4A3}—Homo sapiens (Human)

CA35_HUMAN (P25940) Collagen alpha 3(V) chain precursor. {GENE:COL5A3}—Homo sapiens (Human)

CA36_HUMAN (P12111) Collagen alpha 3(VI) chain precursor. {GENE:COL6A3}—Homo sapiens (Human)

CA39_HUMAN (Q14050) Collagen alpha 3(IX) chain precursor. {GENE:COL9A3}—Homo sapiens (Human)

CA44_HUMAN (P53420) Collagen alpha 4(IV) chain precursor. {GENE:COL4A4}—Homo sapiens (Human)

CA54_HUMAN (P29400) Collagen alpha 5(IV) chain precursor. {GENE:COL4A5}—Homo sapiens (Human)

CA64_HUMAN (Q14031) Collagen alpha 6(IV) chain precursor. {GENE:COL4A6}—Homo sapiens (Human)

EMD2_HUMAN (Q96A83) Collagen alpha 1(XXVI) chain precursor (EMI domaincontaining protein 2) (Emu2 protein) (Emilin and multimerin-domaincontaining protein 2). {GENE: EMID2 OR COL26A1 OR EMU2}—Homo sapiens(Human)

Natural gelatins are known to comprise RGD sequences. It is importanthowever that a collagenous polypeptide molecule does not contain toolarge parts without RGD motifs. Too large parts without RGD sequencereduce the possibility of cell attachment when such a collagenouspolypeptide is used for instance in tissue engineering applications suchas artificial skin. Apparently not all RGD sequences in a collagenouspolypeptide are under all circumstances available for cell attachment.It was found that cell attachment was remarkably improved in collagenouspolypeptides according to the invention compared to gelatins having astretch of amino acids of more than 350 without an RGD sequence. Forcollagenous polypeptides of less than 350 amino acids it is sufficientto have a percentage of RGD sequences of at least 0.4. Note that for acollagenous polypeptide of 251-350 amino acids this means that at least2 RGD motifs are present.

Thus, either fragments enriched in RGD triplets may be identified innatural collagenous proteins, and/or natural collagenous proteins may bemodified generate a polypeptide with a suitable number and distributionof RGD triplets. Nucleic acid sequences encoding suitable polypeptidesmay then be made and expressed in a suitable host cell or organism.

In a preferred embodiment the RGD-enriched collagenous polypeptide isprepared by recombinant DNA technology. Recombinant collagenouspolypeptides of this invention are preferably derived from collagenoussequences. Nucleic acid sequences encoding collagens have been generallydescribed in the art. (See, e.g., Fuller and Boedtker (1981)Biochemistry 20: 996-1006; Sandell et al. (1984) J Biol Chem 259:7826-34; Kohno et al. (1984) J Biol Chem 259: 13668-13673; French et al.(1985) Gene 39: 311-312; Metsaranta et al. (1991) J Biol Chem 266:16862-16869; Metsaranta et al. (1991) Biochim Biophys Acta 1089:241-243; Wood et al. (1987) Gene 61: 225-230; Glumoff et al. (1994)Biochim Biophys Acta 1217: 41-48; Shirai et al. (1998) Matrix Biology17: 85-88; Tromp et al. (1988) Biochem J 253: 919-912; Kuivaniemi et al.(1988) Biochem J 252: 633640; and Ala-Kokko et al. (1989) Biochem J 260:509-516.).

For pharmaceutical and medical uses, recombinant collagenouspolypeptides with amino acid sequences closely related to or identicalto amino acid sequences of natural human collagens are preferred. Aminoacid sequences closely related to human collagens (also referred to asproteins which are “essentially similar” to human collagens) are thosesequences which comprise at least about 50, 60, 70, 75, 80, 90, 95, 98,99% or more amino acid sequence identity over the full length to humancollagen proteins, such as for example the proteins listed above.Sequence identity is determined using pairwise alignment, whereby twopeptide sequences are optimally aligned using programs such as GAP or‘needle’ (the equivalent of GAP provided in EmbossWIN v2.10.0) usingdefault parameters. GAP and “needle” uses the Needleman and Wunschglobal alignment algorithm to align two sequences over their entirelength, maximizing the number of matches and minimizes the number ofgaps. Generally, the GAP default parameters are used, with a gapcreation penalty=50 (nucleotides)/8 (proteins) and gap extensionpenalty=3 (nucleotides)/2 (proteins). Also included are herein fragmentsof human collagen proteins and of essentially similar proteins, such asfragments of at least 30, 50, 100, 150, 200, 250, 300, 350, 400, 500,600, 800, 900, 1000 or more consecutive amino acids. As described below,such fragments may also be used to make repeats thereof, such that thefragment is repeated 2, 3, 4, 5, 10, 15, 20, 30, 50, 70, 80, 90 100times or more. Optionally spacers may be present between the repeats.

More preferably the amino acid sequence of the collagenous polypeptideis designed by a repetitive use of a selected amino acid sequence of ahuman collagen. A part of a natural collagen sequence comprising an RGDmotif is selected. The percentage of RGD motifs in such a selectedsequence depends on the chosen length of the selected sequence,selection of a shorter sequence results in a higher RGD percentage.Repetitive use of a selected amino acid sequence results in a gelatinwith a higher molecular weight, which is non-antigenic and with anincreased number of RGD motifs (compared to natural gelatins orcollagens).

Thus in a preferred embodiment the RGD-enriched collagenous polypeptidecomprises a part of a native human collagen sequence. Preferably theRGD-enriched collagenous polypeptide consists for at least 80% of one ormore parts of one or more native human collagen sequences. Preferablyeach of such parts of human collagen sequences has a length of at least30 amino acids, more preferably at least 45 amino acids, most preferablyat least 60 amino acids, up to e.g. 240, preferably up to 150, mostpreferably up to 120 amino acids, each part preferably containing one ormore RGD sequences. Preferably the RGD-enriched collagenous polypeptideconsists of one or more parts of one or more native human collagensequences.

An example of a suitable source of a collagenous polypeptide forpreparing the films according to this invention is human COL1A1-1. Apart of 250 amino acids comprising an RGD sequence is given in WO04/85473.

RGD sequences in collagenous polypeptides can adhere to specificreceptors on the cell wall called integrins. These integrins differ intheir specificity in recognizing cell binding amino acid sequences.Although both natural gelatin and, for example, fibronectin may containRGD sequences, gelatin can bind cells that will not bind to fibronectinand vice versa. Therefore fibronectin comprising RGD sequences cannotalways replace gelatin for cell adhesion purposes.

The RGD-enriched collagenous polypeptides can be produced by recombinantmethods as disclosed in EP-A-0926543, EP-A-1014176 or WO 01/34646. Forthe production and purification of collagenous polypeptides that aresuited for preparing films of this invention reference is made to theexamples in EP-A-0926543 and EP-A-1014176. The preferred method forproducing an RGD-enriched collagenous polypeptides is by starting with anatural nucleic acid sequence encoding a part of the collagen proteinthat includes an RGD amino acid sequence. By repeating this sequence anRGD-enriched collagenous polypeptide is obtained.

If X-RGD-Y is a part of the natural collagen amino acid sequence, a(part of a) collagenous polypeptide with three RGD amino acid sequenceswould have the structure -X-RGD-Y-(GXYG)m-X-RGD-Y-(GXYG)n-X-RGD-Y-, withm and n being integers starting from 0. By varying n the number of RGDsequences on the total amino acids the percentage of RGD motifs can becontrolled. A clear advantage of this method is that the amino acidsequence remains most natural and thus has the lowest risk of inducingimmunological response in clinical applications.

Starting from a natural nucleic acid sequence encoding (part of) acollagenous polypeptide, also point mutations can be applied so as toyield a sequence encoding an RGD sequence. Based on the known codons apoint mutation can be performed so that an RGX sequence after mutationwill yield an RGD sequence, alternatively also an YGD sequence can bemutated to yield an RGD sequence. Also it is possible to carry out twomutations so that an YGX sequence will give an RGD sequence. Also it maybe possible to insert one or more nucleotides or delete one or morenucleotides giving rise to a desired RGD sequence.

Further it was found that the properties of the gelatin used to make thefilm, in terms of the degree of glycosylation, optionally in combinationwith the number of RGD triplets in the gelatin, can influence the speedof cell growth, the cellular attachment to the film and the cell-to-celladhesion of the autologous cells, whereby the final thickness andquality (cell density and adhesion strength) of the artificial skinlayer can be influenced and improved. Low or no glycosylation of thegelatin used and/or high numbers of RGD triplets in the gelatin used tomake the film had a positive effect on the speed of cell (e.g.fibroblast and keratinocyte) growth on the surfaces, and on the cellularadhesion properties. Thus, it was found that by controlling the ratio ofglycosylation to the number of RGD triplets, films of good quality canbe made.

In a further embodiment, the gelatins used to make the film are low inglycosylation and preferably also substantially pure when used for filmmaking. There are various methods for ensuring that glycosylation is lowor absent. Glycosylation is a posttranslational modification, wherebycarbohydrates are covalently attached to certain amino acids of theprotein or polypeptide. Thus both the amino acid sequence and the hostcell (and enzymes, especially glycosyltransferases, therein) in whichthe amino acid sequence is produced determine the glycosylation pattern.There are two types of glycosylation: N-glycosylation begins withlinking of GlcNAc (N-actylglucosamine) to the amide group of asparagines(N or Asn) and O-glycosylation commonly links GalNAc(N-acetylgalactosamine) to the hydroxyl group of the amino acid serine(S or Ser) or threonine (T or Thr).

Glycosylation can, therefore, be controlled and especially reduced orprevented, by choosing an appropriate expression host, and/or bymodifying or choosing sequences which lack consensus sites recognized bythe hosts glycosyltransferases. Obviously, chemical synthesis ofproteins or polypeptides results in unglycosylated proteins. Also,glycosylated proteins may be treated after production to remove all ormost of the carbohydrates or unglycosylated proteins may be separatedfrom glycosylated proteins using known methods.

In yeasts N-linked glycosylation of asparagine occurs on the consensussites Asn-X-Thr or Asn-X-Ser, wherein X is an amino acid. Commonlyglycosylation in yeast results in N-linked and O-linked oligosaccharidesof mannose. Thus, for expression in yeast the nucleic acid sequence maybe modified or selected so that consensus sites are reduced orpreferably absent. The Asn codon and/or the Thr codon may be modified,e.g. by mutagenesis or de novo synthesis. Preferably Asn and/or Thr isreplaced by another amino acid. Also Asp may be replaced by anotheramino acid. In one embodiment the polypeptide sequence contains no Serand/or no Asn.

To analyse the degree of post-translational modification or to determinethe content of glycosylation mass spectrometry, such as MALDI-TOF-MS(Matrix Assisted Laser Desorption Ionization mass spectrometry) can becarried out as known in the art.

Alternatively the amount of glycosylation can be determined using thetitration method described by Michel Dubois et al, “Colorimetric Methodfor Determination of Sugars and Related Substances”, AnalyticalChemistry, vol 28, No. 3, March 1956, 350 356. This method can be usedto determine simple (mono) sugars, oligosaccharides, polysaccharides,and their derivatives, including the methyl ethers with free orpotentially free reducing groups, and thus the method is quantitative.

The content of glycosylation of the colleganous polypeptide used ispreferably equal to, or less than about 2 (m/m) %, more preferably lessthan about 1 (m/m) %, most preferably less than about 0.5 (m/m) %, 0.2(m/m) % or 0.1 (m/m) %. In a preferred embodiment the glycosylationcontent (or degree of glycosylation) is zero. The content ofglycosylation refers to the total carbohydrate weight per unit weight ofthe collagenous polypeptides as determined by for example MALDI-TOF-MS(Matrix Assisted Laser Desorption Ionization mass spectrometry) orpreferably the titration method by Dubois et al. referred to above. Theterm ‘glycosylation’ refers not only to monosaccharides, but also topolysaccharides such as di-, tri- or tetra saccharides.

In another embodiment the number of RGD triplets (defined as the numberof RGD triplets per 250 amino acids of collagenous polypeptide) ispreferably at least 2, more preferably at least 3, 4, 5, 6, 7, 8 oremore. Such collagenous polypeptides are referred to as “RGD-enrichedcollagenous polypeptides”.

Thus, in one embodiment a method of making a film which has advantageousproperties in the manufacture of artificial skin equivalents isprovided. In one embodiment a film is made using collegenouspolypeptides having low or no glycosylation and/or a high number of RGDtriplets. Film made using the above RGD enriched polypeptides and/orpolypeptides low in glycosylation (including polypeptides with zeroglycosylation) are also provided. Such film are suitably made asdescribed herein.

Thus the collagenous polypeptides can be produced by expression ofnucleic acid sequence encoding such polypeptide by a suitablemicro-organism. The process can suitably be carried out with a fungalcell or a yeast cell. Suitably the host cell is a high expression hostcells like Hansenula, Trichoderma, Aspergillus, Penicillium,Saccharomyces, Kluyveromyces, Neurospora or Pichia. Fungal and yeastcells are preferred to bacteria as they are less susceptible to improperexpression of repetitive sequences. Most preferably the host will nothave a high level of proteases that attack the collagen structureexpressed. In this respect Pichia or Hansenula offers an example of avery suitable expression system. Use of Pichia pastoris as an expressionsystem is disclosed in EP-A-0926543 and EP-A-1014176. The micro-organismmay be free of active post-translational processing mechanism such as inparticular hydroxylation of proline and also hydroxylation of lysine.Alternatively the host system may have an endogenic prolinehydroxylation activity by which the collagenous polypeptide ishydroxylated in a highly effective way. The selection of a suitable hostcell from known industrial enzyme producing fungal host cellsspecifically yeast cells on the basis of the required parametersdescribed herein rendering the host cell suitable for expression ofcollagenous polypeptides which are suitable for use as artificial skinin combination with knowledge regarding the host cells and the sequenceto be expressed will be possible by a person skilled in the art.

In another embodiment the recombinant collagenous polypeptides have ahigher glass transition temperature than natural occurring gelatins.Such sequences are described in WO 05/11740.

The film obtainable using the methods described below has advantageousproperties when it is used to make human artificial skin equivalents,using autologous human cells, especially fibroblasts and/orkeratinocytes. As shown in the examples, the film enables better(optimized, faster) cell growth compared to cell growth on filmcomprising high glycosylation and/or low numbers of RGD triplets. Thismay be due to the enhanced cell-to-film and/or cell-to-cell attachmentproperties, whereby cell spread across the film is improved. A dense,full thickness artificial skin is obtainable by growing cells on atleast one, but preferably both sides of the film. The cells are grownuntil the skin equivalent is preferably at least about 10, 15, 20 μmthick, or more. Preferably the skin equivalent is “full thickness” (atleast about 15-20 μm thick in total). Preferably, multiple layers ofcells (e.g. fibroblasts and/or keratinocytes) are grown on at least one,more preferably both sides of the film. This includes the formation of ahorny layer.

The method of making an artificial skin comprises the steps of

-   -   producing a film comprising an RGD enriched collagenous        polypeptide and/or a polypeptide having a low degree of        glycosylation (e.g. no glycosylation);    -   contacting the film on one or both sides with live or viable        human cells, especially autologous human cells (e.g. fibroblasts        and/or keratinocytes);    -   incubating the film comprising the cells for a suitable period        of time, under suitable conditions for cell growth    -   and optionally repeating the steps of contacting and incubating        one or more times.

Cells that can be grown on at least one side of the non-porous film ofthe invention can be any living, genetically modified or malignantliving cell. Preferably normal (healthy) cells, such as those that occurin the human dermis or epidermis, are cultured on the non-porous film.Preferred are human (or mammalian) cells that occur in skin tissue suchas fibroblasts, keratinocytes, melanocytes, Langerhans'cells, and thelike. In a preferred embodiment the cells are obtained from the subjectto be treated (they are “autologous cells”). In one embodiment theinvention provides a non-porous film comprising on one side fibroblasts.In another embodiment the invention provides a non-porous filmcomprising on one side keratinocytes. Preferably the keratinocytes areexposed to air during culturing so that a horny layer (stratum corneum)is formed at the air-culture interface.

In a preferred embodiment a layer comprising fibroblasts on one side ofthe non-porous film while a layer comprising keratinocytes on theopposite side is provided to avoid that the fibroblasts interfere withkeratinocyte growth and differentiation. By exposing the keratinocyteculture to air the formation of horny tissue, hence a stratum corneum,occurs. Thus a full human skin-equivalent is provided comprising anon-porous film according to the present invention comprising on oneside thereof a layer comprising fibroblasts and on the opposite side ofthe layer comprising fibroblasts a layer comprising keratinocytes. Inparticular the layer comprising keratinocytes comprises at the surfacethat is not in contact with the non-porous film horny tissue.

After culturing, such a material is suitable for use as artificial skinor as a test substrate for medicines or pharmaceutical or cosmeticcompounds, for instance for assessing the permeability of medicines orpharmaceutical or cosmetic compounds through the artificial skin, and/orthe influence on cells on either side of the non-porous collagenouspolypeptide of the artificial skin or testing properties such of forexample UV absorbing compounds. In case of the embodiment having on bothsides of the non-porous film cells, in particular fibroblasts andkeratinocytes, the non-porous crosslinked gelatin film resembles thebasal membrane found in natural skin, thereby providing a full humanskin equivalent closely resembling natural skin. This may be of benefitfor example in particular for effects of test substrates on skin andalso for the treatment of wounds.

Culturing or growing viable or living cells on one or both sides of thefilm can be done using cell culture methods known in the art and asdescribed in the Examples. Nutrients and other components may either beadded together with the cells or separately, and the films comprisingthe cells are incubated for a sufficient period of time and undersuitable conditions for cells growth and/or cells divisions to occur.The non-porous crosslinked gelatin film is particularly suited for theculturing of cells on both sides as the gelatin film has the requiredphysical stability, or mechanical strength, for optimal handling duringculturing, in particular in the step of turning the film upside down forculturing the second layer of cells.

The non-porous film may further comprise one or more bioactive compoundssuch as hormones, growth promoters, antibiotics, immune-suppressors, andthe like. Further the non-porous film may comprise one or more compoundsthat can aid in the wound healing process. A “bioactive compound” is anycompound (either a natural compound or a synthetic compound) whichexerts a biological effect on other cells. Such compounds are widelyavailable in the art. The compound may be incorporated into the filmduring its manufacture or, alternatively, it may be added subsequentlyto one or both sides of the film.

The non-porous film can also be used in cases where skin loss is lessextensive but needs to be replaced still, for example in cases ofchronic open wounds or in the case of bedsores that occur with forexample paralysis.

In another embodiment a method for manufacturing a film according to theinvention is provided. This method comprising the steps of:

a) providing a collagenous polypeptide solution of between 2 and 30weight percent in an aqueous solution,

b) adding a suitable amount of (one or more) crosslinking compound(s) tosaid aqueous solution, preferably between about 0.02 and 5.0 millimol of(one or more) crosslinking compound(s) per gram collagenous polypeptide(or any other suitable amount as described herein above)c) coating said collagenous polypeptide solution onto a substrate thatwas, optionally, first subjected to an adhesion improving treatment ofat most 30 watt·minute per square meterd) drying said coated substrate, and optionallye) separating the dried non-porous film from the substrate.

Also provided is a method for producing a non-porous film suitable forculturing living or viable cells on at least one side thereof comprisingthe steps of:

a) providing a collagenous polypeptide solution of between 2 and 30weight percent in an aqueous solution,

b) coating said collagenous polypeptide solution onto a substrate thatwas, optionally, first subjected to an adhesion improving treatment ofat most 30 watt·minute per square meter

c) drying said coated substrate,

d) subjecting said dried coated substrate to radiation (as describedherein above) to form crosslinks between said collagenous polypeptides,and optionally

e) separating the dried non-porous film from the substrate.

The non-porous film of this invention can be produced efficiently andwith high speed by coating the collagenous polypeptide solution onto asuitable substrate. The coating solution is prepared by dissolvingbetween about 2 and 30 weight percent of (one or more) collagenouspolypeptide(s) in an aqueous solvent. Preferably the concentration ofthe collagenous polypeptide is between about 5 and 20 weight percent,more preferably between about 10 and 15 weight percent. In caserecombinant collagenous polypeptides are used that cannot form stabletriple helixes at room temperature or lower temperature, higherconcentrations can be used than with natural gelatin or collagen. Theaqueous solution contains at least 50 weight percent water, preferablyat least 60 weight percent.

An additional solvent may be added to reduce the surface tension of thecoating solution in order to improve coatability. Suitable solvents arethose that have lower surface tension than water and that in principlecan be removed completely by drying. Suitable solvents are for examplelower alkyl alcohols such as ethanol, ketones such as acetone, loweralkyl acetates such as ethylacetate and the like. Preferred additionalsolvents are lower alkyl alcohols such as methanol, ethanol,(iso)propanol. Preferably ethanol is used. Lower alkyl means that thealkyl chain has from 1 to about 6 carbon atoms.

The coating solution is then coated onto a solid substrate. As a coatingequipment any method known in the art can be used such as slide beadcoating, curtain coating, bar coating, cast coating and the like.

Suitable substrates are substrates having a resin surface such as apolyolefin layer. Preferably the resin layer comprises a polyethylene(PE) or polypropylene (PP), which can be a high density, a low density,a linear low density, a metallocene PE or PP or a mixture thereof. Thesubstrate can also be a paper base coated with a resin layer.

Before coating, the resin surface is optionally subjected to an adhesionpromoting treatment such as a flame treatment, a corona treatment or aplasma treatment is necessary of at least 1.5 watt·minute per squaremeter, preferably at least 2.5 watt·minute per square meter, and at most30 watt·minute per square meter, preferably at most 25, 20, 15, 10 or 5watt·minute per square meter. Purpose of the adhesion promotingtreatment is to provide enough adhesion so that the material can becoated, dried and subjected to processes such as rolling up or cuttingwithout release of the non-porous film. On the other hand the adhesionshould be weak enough to facilitate easy separation from the substrateprior to use for growing or culturing cells.

Just before coating the coating solution onto a substrate, acrosslinking compound(s) may be added. Depending on the desired degreeof biodegradability the amount of crosslinking compound(s) added can bebetween, for example, 0.02 and 0.5 millimol crosslinking compound(s) pergram collagenous polypeptide, between 0.05 and 1 between 0.1 and 2.0,between 0.25 and 2.5, or between 1.0 and 5.0 millimol crosslinkingcompound(s) per gram collagenous polypeptide.

Adding ‘just before coating’ or ‘immediately prior to coating’ meansthat after addition of crosslinking compound the coating solution iscoated onto a substrate before the viscosity increase is too high. Thereaction speed of crosslinking and thus the increase of viscositydepends, amongst other factors, on concentrations of both crosslinkingcompound and collagenous polypeptide. In practical situations thecoating liquid is coated within at most about two hours after addition,preferably within at most about 60 minutes, more preferably within atmost about 30 minutes after addition of the crosslinking compound to thesolution.

Drying can be done by any method known in the art. Preferably the dryingconditions, such as humidity and temperature, are controlled so that toofast drying, resulting in cracking or breaking of the film, isprevented.

Before inoculating or contacting the film with one or more cells, thefilm may optionally be sterilized, for example by exposure to gammaradiation. This can be done before or after peeling (separating) thefilm from the substrate. Alternatively the whole process is carried outunder sterile conditions and by using sterile components, so that thefilm is sterile prior to being contacted with live or viable cells. Thedesired cells may also be contacted with one of the surfaces while thefilm is still attached to the substrate.

In another embodiment the collagenous polypeptides in the film arecrosslinked after coating, by exposure to radiation, such asUV-radiation or electron beam. This can replace the addition ofcrosslinking compound or can be used in combination therewith.

A film obtainable by any of the methods described herein is also anembodiment of the invention.

The film according to the invention may then be contacted with live orviable cells on one and/or both sides of the film. This can be doneusing known methods, for example inoculating the surface with a cellsuspension by pouring or pipetting the (liquid or semi-solid) suspensiononto the surface or by or dipping or laying the film surface into/ontothe cell suspension. The cells may further be distributed on the film'ssurface by streaking or other methods. Further, nutrients and or othercomponents may be supplied to the cells and the films are incubated forsufficient time and under suitable conditions to allow cell growthand/or cell division(s).

DESCRIPTION OF THE FIGURES

FIG. 1: Effect of crosslinking on initial thickness and swelling of anon-porous film. The graph shows the EDC/lysine ratio (tetramer example1, 72.6 kDa) vs dry thickness (μm) and swelling (μm, H₂O 37° C.).

FIG. 2: Effect of crosslinking on degradation speed of a non-porousfilm. The graph shows the degradation speed of EDC cross-linked tetramer(72.6 kDa) to a bacterial collagenase solution with 10 CDU/mg gelatin.

FIG. 3: Diffusion cell for testing permeability of a non-porous film,wherein 1=donor compound, 2=receptor compartment, 3=receptor input,4=compound and receptor output for analysis, 5=⅛″ OD× 1/32″ wall tubing.

FIG. 4: Effect of crosslinking on permeability and degradation of anon-porous film. The graph shows the permeability for lysozyme (14.3kDa, 1st fraction) and degradation (weight remaining) after 25 h of EDCcross-linked tetramer (72.6 kDa) to a collagenase solution with 10CDU/mg gelatin.

EXAMPLES Preparation of a Non-Porous Film or Film of this Invention

A natural gelatin or a recombinantly produced collagenous polypeptide,as described for example in EP-A-1398324, of a molecular weight up to100 kilo Dalton is dissolved in demineralized water at a temperature of40° C. After the polypeptide is dissolved the temperature is increasedto 60 degrees Celsius for 30 minutes to fully uncurl the gelatin orcollagen strands, after which the temperature is decreased again to 40degrees Celsius. To improve wettability 15-30% (weight/weight) 96% pureEtOH is added to obtain final collagenous polypeptide concentrations of10-25% (weight/weight).

Depending on the cross-linking compound the pH is adjusted with 1M NaOHor 1M HCl to 5-6 when using glutaraldehyde (GTA: 25% solution(weight/weight)) and to 7-8 when using N-Ethyl-N′-(3-Dimethylaminopropyl)carbodiimide.HCl (EDC: 25% solution (weight/weight)). Thecrosslinking compound is added to the gelatin solution just beforecoating, that is, before viscosity due to crosslinking becomes too high.

The collagenous polypeptide solution is thoroughly mixed with thecrosslinking compound solution and directly after mixing coated on apolyethylene substrate. Wet coating thickness of 100-400 μm is appliedwhich after drying results in dry membrane thickness of 10-100 μm.

Drying may be done for example at ambient conditions for at least 24 h.

After drying, films are cut and irradiated with gamma rays at a doses ofat least 25 kGy to realize sterile gelatin films

Culture of Keratinocytes and Fibroblasts:

Keratinocytes and fibroblasts were isolated from normal human skinobtained from breast surgery. Keratinocytes were grown in keratinocytemedium using 3 parts of Dulbecco's modified eagles medium (DMEM) and 1part of Ham's F12 medium supplemented with 5% serum (fetal calf) andvarious other additives e.g 100 microgram streptomycin/ml and 100 I.U.penicillin/ml. For establishment of human skin equivalents keratinocytesof passage 2 were used. Fibroblasts were grown in DMEM, supplementedwith 5% calf serum. For fabrication of skin equivalents fibroblasts ofpassage 2-9 were used. Keratinocytes and fibroblasts were grown toconfluence in plastic tissue culture dishes.

Preparation Method of Human Skin Equivalents:

The collagenous polypeptide films were washed during 24 hours inbuffered saline solution at room temperature. After 1 and 2 hours, thebuffered saline solution was refreshed. After washing, fibroblasts wereseeded onto the films and either

-   a. incubated for 3 days in fibroblast medium with 5% serum, 1    nanogram/ml Epidermal Growth Factor (EGF) and various other    additives.-   b. incubated for 3 days in fibroblast medium with 1% serum, 1    nanogram/ml EGF and various other additives. During this culture    period—at day two—keratinocytes were seeded onto the backside of the    collagenous polypeptide films.

Human skin equivalents are grown onto metal grid supports.

After 3 days, the combined keratinocyte/fibroblast cultures were liftedto the air-liquid interface and cultured in DMEM/Ham's F12 mediumsupplemented with 1 nanogram/ml EGF, in the absence of serum. Cells weregrown for an additional 10 days to confluence.

Example 1 Preparation of Nonporous Film from a Recombinantly ProducedTetramer of 72.6 Kilo Dalton as Described in EP-A-1398324

Totally 11.4 g of the tetramer was dissolved in 34.2 g demineralizedwater at 40 degrees Celsius. After dissolving, the temperature wasincreased to 60 degrees Celsius for 30 minutes and then decreased againto 40 degrees Celsius. Additional 11.4 g EtOH (96% pure) was added. pHof the solution was adjusted with 1M NaOH to 7.5.

25% EDC(N-Ethyl-N′-(3-Dimethyl aminopropyl)carbodiimide.HCl) solutionwas prepared by dissolving 1 g EDC in 3 g demineralized water.

Additions of the crosslinking compound solution to the collagenouspolypeptide solutions were done according table 1. After addition themixtures were stirred thoroughly and the mixtures were applied onnon-treated photographic base-paper with a polyethylene top layer. Witha spirally wound ‘Large K Hand Coater Bar’ No. 125 a wet film deposit of125 μm was coated on A4 sized substrates. The coated films were left todry at room temperature for at least 24 h.

Dry thickness was measured using a Lorentzen & Wettre micrometer type221. Swelling can be measured with a method as described by Flynn andLevine (Photogr. Sci. Eng., 8, 275 (1964). Physical strength wasdetermined qualitatively by manually handling a film. The designationmeans too weak, ‘+’ no tear during normal handling, ‘+/−’ means that inabout 50 percent of the tests the film tore and ‘++’ means no tear evenafter applying more force than necessary. Brittleness was also testedqualitatively in a similar manner by bending the film. Results of drythickness, water swelling (vertical) and physical properties are alsolisted in table 1:

TABLE 1 EDC/Lysine ratio Dry thickness Swelling Physical Solution(mol/mol) (micron) (micron) strength brittleness I 0.4 25 159 +/− + II0.5 24 138 + + III 0.6 22 133 +/++ + IV 1.0 23 64 ++ + V 2.0 21 54 − −

See FIG. 1 for the graphic processing of the swelling data.

The obtained films were cut to circular membranes with a diameter of 27millimeter and were sterilized by means of gamma irradiation with adoses of at least 25 kGy.

Preparation Method of Full Human Skin Equivalent:

-   -   A gelatin membrane was placed in a petridish (using a pair of        tweezers);    -   Phosphate buffered saline solution was added to the petridish        for washing the membrane (at RT). After 2 hrs. the PBS solution        was removed by a pipette and fresh PBS solution was added to the        membrane;    -   After 1 day PBS solution was removed (by pipette) and fibroblast        culture medium was added for an additional washing step;    -   After 2 hrs culture medium solution was removed by pipette and        fibroblasts were seed on the membrane;    -   The cells were allowed to attach to the membrane for ˜1 h;    -   The fibroblast culturing medium was added in such an amount that        the membrane was not air-exposed since fibroblasts have to grow        under wet conditions;    -   Culturing took place for 1 week, 2 times per week culture medium        was refreshed (removal by pipette);    -   Membrane with fibroblasts was turned upside down using a        Millipore filter (TETP02500, 8 μm). The membrane was lifted via        a pair of tweezers and put upside down on a metal grid;    -   The medium was changed to keratinocyte medium as described        above;    -   Keratinocytes were seeded on the backside of the gelatin        membrane in a metal ring (to prevent dispersion of the cells).        After adherence of the keratinocytes the metal ring was removed        and the culture continued for another 7 days;    -   After 7 days, the cultures were exposed to the air interface and        cultured for another 14 days; culture medium was refreshed 2        times per week); no extra handlings were needed since the amount        of medium was reduced so that the keratinocytes remained above        the liquid surface.        Evaluation of Full Human Skin Equivalent:    -   skin equivalents were harvested and fixed in 4%        paraformaldehyde, dehydrated and embedded in paraffine; then the        embedded tissue was cut into slices and stained with        haematoxyline/eosine;    -   Stained slices were visualized by light microscope;    -   Clearly several layers (multilayer) of keratinovcytes were        visible having on the air-exposed side thereof horny tissue        (stratum corneum); fibroblasts were separated from the        keratinocytes by the gelatin membrane.

Example 2 In Vitro Degradation of the Cross-Linked Membranes

Bacterial collagenase (activity of >125 CDU/mg (One Collagen DigestionUnit liberates peptides from collagen equivalent in ninhydrin color to1.0 μmole of leucine in 5 hr at pH 7.4 at 37° C. in the presence ofcalcium ions)) from Clostridium histolyticum (Sigma-Aldrich, EC3.4.24.3) was selected as an enzyme for the degradation studies becauseof its specificity for collagen. These collagenase preparations containat least six different collagenases which are capable of cleavingpeptide bonds within the triple helical structure and have a specificityfor the Pro-X-Gly-Pro-Y region, splitting between X and Gly.

In a typical degradation experiment, a 10 milligram sample ofcross-linked collagenous polypeptide with either GTA or EDC is immersedin 0.5 ml of a 0.1 M Tris-HCl buffer solution (pH 7.4) containing 0.005MCaCl₂ and 0.05 mg/ml sodium azide and incubated at 37° C. After onehour, 0.5 ml collagenase solution in Tris-HCl buffer (37° C.) was addedto give the desired final concentration and absolute amount ofcollagenase (100 CDU/ml or 10 CDU/mg collagenous polypeptide).

The degradation was discontinued at the desired time interval by theaddition of 0.1 ml 0.25M EDTA (Titriplex III) and cooling of the system.

The weight-loss of the cross-linked collagenous polypeptide samplesduring the degradation was determined by a gravimetrical method.

Samples were dried overnight under vacuum over KOH and were weighted.Thereafter the samples were degraded as described above. After apre-determined degradation period, EDTA was added and the tubes werecentrifuged at 600G for 10 minutes and the remaining solution wasdiscarded. The resulting pellet was washed with distilled water andcentrifuged. This washing procedure was conducted three times in total.After the final washing step, the remaining pellet was freeze dried andweighted to determine the weight-loss of the collagenous polypeptidesamples.

See FIG. 2 for the graphical presentation of the data.

Example 3 Permeability and Degradation Level vs. Cross-Link Density

To determine the permeability of the prepared membranes a diffusionexperiment was initiated. 193 mg of Lysozyme, a globular protein of 14.3kDa, was dissolved in 5 ml physiological salt solution to obtain alysozyme concentration of 38.6 mg/ml (donor solution).

The EDC cross-linked gelatin membranes with a cross-linking density of0.4, 0.6, 1.0 and 1.5 EDC/lysine (mol/mol) were mounted in the diffusioncells (see FIG. 3) and 300 μl of the donor solution was put on top ofthe mounted membranes.

The receptor fluid (also physiological salt solution) flow is 1 ml/hr.During the whole experiment the temperature of the system was kept at37° C.

The first fraction of 5 ml and the diluted donor solution were analyzedby means of GPC at a wavelength of 280 nm.

The results were compared to the degradation level (weight remaining %)after 25 h. See FIG. 4 for the graphical presentation of the data whichshow that both parameters, permeability and degradation, can becontrolled by the cross-linking density.

Example 4 Effect of RGD Triplets and/or Glycosylation of the CollagenousPolypeptide on Cell Growth

Method:

To test glycosylation and/or RGD triplet numbers on human cell growth,various polypeptides were made.

Results:

Glycosylation (m/m) % Nr. of RGD triplets Cell growth 7-9 0 Very bad 1-20 bad 1-2 1 moderate ≦1-2   5 Very good

1. A non-porous film having an average pore size of less than 1 μm asdetermined by scanning electron microscope, comprising on at least oneside thereof a layer comprising living or viable cells, wherein thenon-porous film comprises a collagen polypeptide comprising at least oneGXY domain having a length of at least 5 consecutive GXY triplets,wherein X and Y each represent any amino acid and wherein at least 20%of the amino acids of said collagen polypeptide are present in the formof consecutive GXY triplets and wherein the film thickness when placedin demineralized water of 37° C. for 24 hours is at most 10 times itsinitial thickness, and wherein said film is crosslinked by: (i) addingbetween 0.02 mmol and 5.0 mmol of a crosslinking compound per gramcollagenous polypeptide; or (ii) exposure to radiation so that thedegree of crosslinking is the equivalent of adding between 0.02 mmol and5.0 mmol of a crosslinking compound per gram collagenous polypeptide. 2.The non-porous film according to claim 1, wherein said collagenpolypeptide is selected from the group consisting of gelatin, naturalcollagen, modified collagen, synthetic collagen, and recombinantcollagen.
 3. The non-porous film according to claim 1, wherein thecollagen polypeptide has at least 0.4 percent RGD motifs.
 4. Thenon-porous film according to claim 1, wherein the collagen polypeptidehas a content of glycosylation of less than or equal to 2 (m/m) %. 5.The non-porous film according to claim 1, wherein the degree ofcrosslinking is the equivalent of adding between 0.5 millimol and 2.0millimol of a crosslinking compound per gram collagen polypeptide. 6.The non-porous film according to claim 1, wherein said crosslinkingcompound is selected from the group consisting of glutaraldehyde,water-soluble carbodiimides, bisepoxy compounds, formalin and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
 7. The non-porous filmaccording to claim 1, wherein said film further comprises one or morebioactive compounds.
 8. The non-porous film according to claim 7,wherein said one or more bioactive compounds are selected from the groupconsisting of a hormone, a growth promoter, an antibiotic and animmune-suppressor.
 9. The non-porous film according to claim 1, whereinsaid film comprises on one side a layer comprising fibroblasts and onthe opposite side a layer comprising keratinocytes.
 10. The non-porousfilm according to claim 9, wherein the layer comprising keratinocytescomprises, at the surface that is not in contact with the non-porousfilm, horny tissue.
 11. The non-porous film according to claim 2,wherein said crosslinking compound is selected from the group consistingof glutaraldehyde, water-soluble carbodiimides, bisepoxy compounds,formalin and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
 12. A methodof treating skin wounds comprising administering to the wound anon-porous film according to claim 1.